Fed-batch cultivation of Saccharomyces cerevisiae in a hyperbaric bioreactor

Fed-batch is the dominating mode of operation in high-cell-density cultures of Saccharomyces cerevisae in processes such as the production of baker's yeast and recombinant proteins, where the high oxygen demand of these cultures makes its supply an important and difficult task. The aim of this work was to study the use of hyperbaric air for oxygen mass transfer improvement on S. cerevisiae fed-batch cultivation. The effects of increased air pressure up to 1.5 MPa on cell behavior were investigated. The effects of oxygen and carbon dioxide were dissociated from the effects of total pressure by the use of pure oxygen and gas mixtures enriched with CO(2). Fed-batch experiments were performed in a stirred tank reactor with a 600 mL stainless steel vessel. An exponential feeding profile at dilution rates up to 0.1 h(-)(1) was used in order to ensure a subcritical flux of substrate and, consequently, to prevent ethanol formation due to glucose excess. The ethanol production observed at atmospheric pressure was reduced by the bioreactor pressurization up to 1.0 MPa. The maximum biomass yield, 0.5 g g(-)(1) (cell mass produced per mass of glucose consumed) was attained whenever pressure was increased gradually through time. This demonstrates the adaptive behavior of the cells to the hyperbaric conditions. This work proved that hyperbaric air up to 1.0 MPa (0.2 MPa of oxygen partial pressure) could be applied to S. cerevisiae cultivation under low glucose flux. Above that critical oxygen partial pressure value, i.e., for oxygen pressures of 0.32 and 0.5 MPa, a drastic cell growth inhibition and viability loss were observed. The increase of carbon dioxide partial pressure in the gas mixture up to 48 kPa slightly decreased the overall cell mass yield but had negligible effects on cell viability.     

Effect of PO2 on growth and physiological characteristics of Candida utilis in a multistage tower fermentor

The effect of increasing the partial pressure of oxygen in the aeration gas on growth and physiological activity of the yeast Candida utilis in a multistage tower fermentor was studied. The measurements were made at steady states of continuous culture for single values of dilution rate, temperature, and pH in all stages of the fermentor and with one given ethanol concentration in the growth medium feed. The partial pressure of oxygen in the gas phase was changed in the range from 165 to 310 torr. The results revealed the existence of the upper critical value of the partial oxygen pressure in the gas phase. It was demonstrated that the upper critical value of PO ₂ influences not only the growth rate, biomass yield, and productivity but also the cell physiology resulting in changes of respiration activity and activity of alcohol and aldehyde dehydrogenases.     

Studies on growth and metabolism of Oenococcus oeni on sugars and sugar mixtures

AIMS: To study the effect of sugars and sugar mixtures on the growth kinetics of Oenococcus oeni NCIMB 11648 in batch culture with the aim of producing a high cell productivity system for starter cultures. METHODS AND RESULTS: The growth of O. oeni was investigated on single sugars (glucose, fructose or sucrose) and their mixtures (glucose-fructose, glucose-sucrose or fructose-sucrose). Better growth was obtained on sugar mixtures compared with growth on a single sugar. The production system of O. oeni biomass was investigated in batch culture with or without pH control with respect to kinetics, specific growth rate and biomass yield. The effect of pH and substrate concentration on fermentation balances and ATP yield were determined. The optimal growth of O. oeni was achieved on the glucose-fructose mixture (9 g l(-1), 1 : 1) at pH 4.5 and 25 degrees C with pH control, with highest cell volumetric productivity (7.9 mg cell l(-1) h(-1)), biomass yield (0.041 g cell g(-1) sugar) and specific growth rate (0.066 h(-1)). CONCLUSIONS: The limitations to the growth of O. oeni were pH and inhibition by end product resulting in poor utilization of the medium with low cell yields. The cell productivity of the system can be improved by the appropriate use of mixed sugar growth medium. SIGNIFICANCE AND IMPACT OF THE STUDY: This study uniquely showed that appropriate sugar mixtures with the correct environmental conditions can significantly improve the productivity of O. oeni cultures.     

The hydrolysis of esters of N-hippurylglycine and N-pivaloylglycine by carboxypeptidase A

The kinetics of the hydrolysis of five esters of N-hippurylglycine (C6H5CONHCH2CONHCH2CO2CRR1CO2H (2 approximately) and seven esters of N-pivaloylglycine ((CH3)3CCONHCH2CRR1CO2H (3 approximately)) by bovine pancreatic carboxypeptidase A (Peptidyl-L-amino-acidhydrolase, EC 3.4.12.2) have been studied at pH 7.5, 25 degrees C and ionic strength 0.5. All N-hippurylglycine esters (2: R=H, R1=H, C2H5, 4-ClC6H4, C6H5CH2) display Michaelis-Menten kinetics up to at least 0.1 M substrate. The N-pivaloylglycine esters display either Michaelis-Menten kinetics (3 approximately: R=H, R1=H, C2H5 C6H5), substrate activation (3 approximately: R=H, R1=4-ClC6H4; R=R1=CH3) or substrate inhibition (3 approximately: R=H, R1=(CH3)2CHCH2, C6H5CH2). Kinetic parameters have been evaluated for each ester and compared with those for the corresponding hippuric acid esters (1 approximately). The enzymic specificity is shown to be identical for the alcohol moieties of the esters 1 approximately, 2 approximately and 3 approximately and unrelated to the occurrence of substrate activation or inhibition phenomena. These latter phenomena are shown to be characteristic of the enzymic hydrolysis of N-acyl amino acid esters but unimportant for N-acyl dipeptide ester substrates.     

Microbial production of poly-D-3-hydroxybutyrate from CO2

This short review covers the biotechnological aspects of the production of poly-D-3-hydroxybutyric acid, P(3HB), from H2, O2 and CO2 by autotrophic culture of the hydrogen-oxidizing bacterium, Ralstonia eutropha. Considering the efficiency of utilization of a gas mixture as substrate, a practical fermentation process using R. eutropha for the mass production of P(3HB) from CO2 should be designed on the basis of a recycled-gas, closed-circuit culture system. Also, maintaining the O2 concentration in a gas phase lower than 6.9% (v/v) is essential to prevent the gas mixture from exploding. Our study, using an explosion-proof fermentation bench plant and a two-stage culture system with a newly designed air-lift fermenter, demonstrated that very high P(3HB) yield and productivity could be obtained while the O2 concentration was maintained below 6.9%. However, a study on the continuous production of P(3HB) from CO2 by chemostat culture of R. eutropha revealed that the productivity and content of P(3HB) in the cells was considerably lower than by fed-batch culture. It is deduced that the use of the hydrogen-oxidizing bacterium, Alcaligenes latus, which accumulates P(3HB) even in the exponential growth phase, will be useful for the effective production of P(3HB) from CO2.     

Measurement and interpretation of the oxygen isotope composition of carbon dioxide respired by leaves in the dark

We measured the oxygen isotope composition (delta(18)O) of CO(2) respired by Ricinus communis leaves in the dark. Experiments were conducted at low CO(2) partial pressure and at normal atmospheric CO(2) partial pressure. Across both experiments, the delta(18)O of dark-respired CO(2) (delta(R)) ranged from 44 per thousand to 324 per thousand (Vienna Standard Mean Ocean Water scale). This seemingly implausible range of values reflects the large flux of CO(2) that diffuses into leaves, equilibrates with leaf water via the catalytic activity of carbonic anhydrase, then diffuses out of the leaf, leaving the net CO(2) efflux rate unaltered. The impact of this process on delta(R) is modulated by the delta(18)O difference between CO(2) inside the leaf and in the air, and by variation in the CO(2) partial pressure inside the leaf relative to that in the air. We developed theoretical equations to calculate delta(18)O of CO(2) in leaf chloroplasts (delta(c)), the assumed location of carbonic anhydrase activity, during dark respiration. Their application led to sensible estimates of delta(c), suggesting that the theory adequately accounted for the labeling of CO(2) by leaf water in excess of that expected from the net CO(2) efflux. The delta(c) values were strongly correlated with delta(18)O of water at the evaporative sites within leaves. We estimated that approximately 80% of CO(2) in chloroplasts had completely exchanged oxygen atoms with chloroplast water during dark respiration, whereas approximately 100% had exchanged during photosynthesis. Incorporation of the delta(18)O of leaf dark respiration into ecosystem and global scale models of C(18)OO dynamics could affect model outputs and their interpretation.     

Cerebral white matter blood flow is constant during human non-rapid eye movement sleep: a positron emission tomographic study.

This study aimed to identify brain regions with the least decreased cerebral blood flow (CBF) and their relationship to physiological parameters during human non-rapid eye movement (NREM) sleep. Using [(15)O]H(2)O positron emission tomography, CBF was measured for nine normal young adults during nighttime. As NREM sleep progressed, mean arterial blood pressure and whole brain mean CBF decreased significantly; arterial partial pressure of CO(2) and, selectively, relative CBF of the cerebral white matter increased significantly. Absolute CBF remained constant in the cerebral white matter, registering 25.9 +/- 3.8 during wakefulness, 25.8 +/- 3.3 during light NREM sleep, and 26.9 +/- 3.0 (ml.100 g(-1).min(-1)) during deep NREM sleep (P = 0.592), and in the occipital cortex (P = 0.611). The regression slope of the absolute CBF significantly differed with respect to arterial partial pressure of CO(2) between the cerebral white matter (slope 0.054, R = - 0.04) and frontoparietal association cortex (slope - 0.776, R = - 0.31) (P = 0.005) or thalamus (slope - 1.933, R = - 0.47) (P = 0.004) and between the occipital cortex (slope 0.084, R = 0.06) and frontoparietal association cortex (P = 0.021) or thalamus (P < 0.001), and, with respect to mean arterial blood pressure, between the cerebral white matter (slope - 0.067, R = - 0.10) and thalamus (slope 0.637, R = 0.31) (P = 0.044). The cerebral white matter CBF keeps constant during NREM sleep as well as the occipital cortical CBF, and may be specifically regulated by both CO(2) vasoreactivity and pressure autoregulation.     

Molar Growth Yields as Evidence for Oxidative Phosphorylation in Streptococcus faecalis Strain 10C1

During the aerobic growth of Streptococcus faecalis strain 10C1, with limiting levels of glucose as the substrate, a molar growth yield (Y) of 58.2 g (dry weight) per mole of glucose was obtained. Under these conditions of growth, glucose was dissimilated primarily to acetate and CO(2). The incorporation of (14)C-glucose into cell material was no greater under aerobic conditions than during anaerobic growth. Assuming an adenosine triphosphate coefficient of 10.5, the aerobic Y cannot be explained solely on the basis of substrate phosphorylation and would appear to substantiate previous enzymatic evidence for oxidative phosphorylation in this cytochromeless species. With mannitol as the substrate, an aerobic Y of 64.6 was obtained. Extracts of mannitol-grown cells contained a nicotinamide adenine dinucleotide (NAD)-linked mannitol-1-phosphate (M-1-P) dehydrogenase. The difference in aerobic Y values with mannitol and glucose as substrates would indicate that the in vivo P/O ratio from the oxidation of reduced NAD generated by the oxidation of M-1-P approximates 0.6. The Y values with pyruvate and glycerol as substrates under aerobic conditions were 15.5 and 24.7, respectively.     

Influence of CO2 and pH on the attachment response of the cercaria of Diplostomum spathaceum (trematoda) (author's transl)]

1. The attachment of the cercaria to artificial substrates (offered via dialyzing membranes) in definite media was investigated under conditions of variable pH and [CO2]. 2. A decrease of the pH of the substrate releases only attachments in CO2 containing media and consequently acts via CO2 systems of the medium. 3. As effective components of CO2 systems, dissolved CO2 + H2CO3 are confirmed. 4. The sensitivity of the reaction on gradients of the CO2 partial pressure (in solution) could be established by offering substrates with lowered pH in CO2 containing media. Thus, by raising the CO2 partial pressure from ca. 0,04% to 0,15% maximal fixation rates were obtained (Fig. 3). 5. The carboanhydrase inhibitor acetazolamide, when added to the medium, had no direct influence on the CO2 receptors.     

重组巴氏毕赤酵母恒化培养动力学及代谢迁移特性研究

通过对甲醇营养型毕赤酵母基因工程菌以碳源甘油为限制性基质进行恒化培养动力学试验 ,结果认为 :(1 )细胞光密度与其干、湿重呈线性关系 ,当细胞光密度 (OD60 0 )为 1 0 0时细胞湿重 (WCW)为 1 2 8 3g L ,细胞干重 (WDW)则为 2 2 9g L ;(2 )基因工程菌P .pastoris的生长与限制性基质甘油残留浓度的关系符合Monod关系式 ,通过 1 μ对 1 S进行线性回归得 μmax=0 .366h- 1,Ks=0 .1 82 3g L ,经参数推导甘油最大菌体得率系数YG =0 .54g g ,菌体维持生长消耗底物系数m =0 .0 0 69g (g·h) ;氧最大菌体系数YX O2 =30 .96g moL ,菌体维持生长时消耗氧系数mO2 =0 .0 0 0 8mol (g·h) ,最适理论稀释速率Dm =0 .341h- 1;(3)从氨水的消耗速率和呼吸商 (RQ)的变化认为随着比生长速率 (μ)的增大 ,甘油代谢流从糖原异生和磷酸戊糖途径线性地向糖酵解和三羧酸循环途径进行代谢迁移 ,即糖酵解和三羧酸循环途径的代谢流量在线性地增大     

Fermentative capacity of baker's yeast exposed to hyperbaric stress

Baker's yeast suspensions were incubated at different pressures (from 1 bar to 6 bar) and different gases [air, O(2) and a mixture of 8% (v/v) CO(2), 21% O(2) and N(2)]. Raising the air pressure from 1 bar to 6 bar stimulated cell growth but had no effect on leavening ability or viability of the cells. A 50% reduction of the CO(2) produced in dough occurred with 6 bar O(2) which also stopped growth. The fermentative capacity of the cells was stimulated by the cells exposure to increased CO(2) partial pressure up to 0.48 bar.     

Carbon monoxide actuates O(2)-limited heme degradation in the rat brain

The biochemical paradigm for carbon monoxide (CO) is driven by the century-old Warburg hypothesis: CO alters O(2)-dependent functions by binding heme proteins in competitive relation to 1/oxygen partial pressure (PO(2)). High PO(2) thus hastens CO elimination and toxicity resolution, but with more O(2), CO-exposed tissues paradoxically experience less oxidative stress. To help resolve this paradox we tested the Warburg hypothesis using a highly sensitive gas-reduction method to track CO uptake and elimination in brain, heart, and skeletal muscle in situ during and after exogenous CO administration. We found that CO administration does increase tissue CO concentration, but not in strict relation to 1/PO(2). Tissue gas uptake and elimination lag behind blood CO as predicted, but 1/PO(2) vs. [CO] fails even at hyperbaric PO(2). Mechanistically, we established in the brain that cytosol heme concentration increases 10-fold after CO exposure, which sustains intracellular CO content by providing substrate for heme oxygenase (HO) activated after hypoxia when O(2) is resupplied to cells rich in reduced pyridine nucleotides. We further demonstrate by analysis of CO production rates that this heme stress is not due to HO inhibition and that heme accumulation is facilitated by low brain PO(2). The latter becomes rate limiting for HO activity even at physiological PO(2), and the heme stress leads to doubling of brain HO-1 protein. We thus reveal novel biochemical actions of both CO and O(2) that must be accounted for when evaluating oxidative stress and biological signaling by these gases.     

Alternative Function of the Electron Transport System in Azotobacter vinelandii: Removal of Excess Reductant by the Cytochrome d Pathway

The N(inf2)-fixing bacterium Azotobacter vinelandii was grown in an O(inf2)-regulated chemostat with glucose or galactose as substrate. Increasing the O(inf2) partial pressure resulted in identical synthesis of the noncoupled cytochrome d terminal oxidase, which is consistent with the hypothesis that A. vinelandii uses high rates of respiration to protect the nitrogenase from oxygen. However, cell growth on glucose showed a lower yield of biomass, higher glycolytic rate, higher respiratory rate, and lower cytochrome o content than cell growth on galactose. Elemental analysis indicated no appreciable change in the C-to-N ratio of cell cultures, suggesting that the major composition of the cell was not influenced by the carbon source. A poor coordination of glucose and nitrogen metabolisms in A. vinelandii was suggested. The rapid hydrolysis of glucose resulted in carbonaceous accumulation in cells. Thus, Azotobacter species must induce a futile electron transport to protect cells from the high rates of glucose uptake and glycolysis.     

Hydrogen-dependent oxygen reduction by homoacetogenic bacteria isolated from termite guts

Although homoacetogenic bacteria are generally considered to be obligate anaerobes, they colonize the intestinal tracts of termites and other environments that are not entirely anoxic in space or time. In this study, we investigated how homoacetogenic bacteria isolated from the hindguts of various termites respond to the presence of molecular oxygen. All strains investigated formed growth bands in oxygen gradient agar tubes under a headspace of H(2)-CO(2). The position of the bands coincided with the oxic-anoxic interface and depended on the O(2) partial pressure in the headspace; the position of the bands relative to the meniscus remained stable for more than 1 month. Experiments with dense cell suspensions, performed with Clark-type O(2) and H(2) electrodes, revealed a large capacity for H(2)-dependent oxygen reduction in Sporomusa termitida and Sporomusa sp. strain TmAO3 (149 and 826 nmol min(-1) mg of protein(-1), respectively). Both strains also reduced O(2) with endogenous reductants, albeit at lower rates. Only in Acetonema longum did the basal rates exceed the H(2)-dependent rates considerably (181 versus 28 nmol min(-1) mg of protein)(-1)). Addition of organic substrates did not stimulate O(2) consumption in any of the strains. Nevertheless, reductive acetogenesis by cell suspensions of strain TmAO3 was inhibited even at the lowest O(2) fluxes, and growth in nonreduced medium occurred only after the bacteria had rendered the medium anoxic. Similar results were obtained with Acetobacterium woodii, suggesting that the results are not unique to the strains isolated from termites. We concluded that because of their tolerance to temporary exposure to O(2) at low partial pressures (up to 1.5 kPa in the case of strain TmAO3) and because of their large capacity for O(2) reduction, homoacetogens can reestablish conditions favorable for growth by actively removing oxygen from their environment.     

Growth of Seliberia carboxydohydrogena carboxy bacteria with an altered composition of the gas mixture

The growth and gas exchange of Seliberia carboxydohydrogena Z-1062 were studied in the regime of turbidostat when the conditions of gaseous nutrition were changed: a decrease in hydrogen concentration and an increase in carbon monoxide concentration, growth on two carbon sources (CO+CO2) and on two energy sources (H2+CO). The inhibition of the bacterial growth by CO was expressed in a decrease of the specific growth rate and in the reduced effectiveness of using a gaseous substrate. When the concentration of carbon monoxide was elevated from 0 to 40% and that of hydrogen was reduced from 80 to 40%, the specific growth rate of the cells was decreased from 0.4 to 0.04 h-1; here, the economic coefficient in terms of hydrogen fell from 3.6 to 0.62 g/g. The CO-oxidizing system of the bacterium was shown to be resistant. The rate of CO oxidation by the culture was from 0.6 to 0.8 L/h per 1 g of the synthesized biomass at the following concentration of gases in the medium (%); H2, 80-40; CO2, 5; O2, 15; CO, 10-40. The rate of CO oxidation by the culture rose when hydrogen concentration was decreased and CO concentration was increased.     

High-Pressure Equipment for Growing Methanogenic Microorganisms on Gaseous Substrates at High Temperature

High-pressure, high-temperature investigations on thermophilic microorganisms that grow on hydrogen or other gaseous substrates require instrumentation which provides sufficient substrate for cell proliferation up to 2 × 10⁸ to 3 × 10⁸ cells per ml under isothermal and isobaric conditions. To minimize H₂ leakage and to optimize reproducibility at high pressure and high temperature, 10-ml nickel tubes with a liquid/gas ratio of 1:2 were used in a set of autoclaves connected in series. By applying a hydraulic pump and a 2.5-kW heating device, fast changes in temperature (up to 400°C) and pressure (up to 400 MPa) can be accomplished within less than 10 min. To quantify bacterial growth, determinations of cell numbers per unit volume yielded optimum accuracy. Preliminary experiments with the thermophilic, methanogenic archaebacterium Methanococcus thermolithotrophicus showed that bacterial growth depends on both temperature and pressure. At the optimum temperature, increased hydrostatic pressure up to 50 MPa enhanced the growth yield; at a pressure of >75 MPa, cell lysis dominated. Changes in cell proliferation were accompanied by changes in morphology.     

Cultivation of microplantlets derived from the marine red alga Agardhiella subulata in a stirred tank photobioreactor

Macrophytic marine red algae are a diverse source of bioactive natural compounds. "Microplantlet" suspension cultures established from red algae are potential platforms for biosynthesis of these compounds, provided suitable bioreactor configurations for mass culture can be identified. The stirred tank bioreactor offers high rates of gas-liquid mass transfer, which may facilitate the delivery of the CO(2) in the aeration gas to the phototrophic microplantlet suspension culture. Therefore, the effects of impeller speed and CO(2) delivery on the long-term production of microplantlet biomass of the model red alga Agardhiella subulata was studied within a stirred tank photobioreactor equipped with a paddle blade impeller (D(i)/D(T) = 0.5). Nutrient medium replacement was required for sustained biomass production, and the biomass yield coefficient based on nitrate consumption was 1.08 +/- 0.09 g dry biomass per mmol N consumed. Biomass production went through two exponential phases of growth, followed by a CO(2) delivery limited growth phase. The CO(2)-limited growth phase was observed only if the specific growth rate in the second exponential phase of growth was at least 0.03 day(-)(1), the CO(2) delivery rate was less than 0.258 mmol CO(2) L(-)(1) culture h(-)(1), and the plantlet density was at least 10 g fresh mass L(-)(1). Increasing the aeration gas CO(2) partial pressure from 0.00035 to 0.0072 atm decreased the cultivation pH from 8.8 to 7.8, prolonged the second exponential phase of growth by increasing the CO(2) delivery rate, and also increased the photosynthetic oxygen evolution rate. Impeller speeds ranging from 60 to 250 rpm, which generated average shear rates of 2-10 s(-)(1), did not have a significant effect on biomass production rate. However, microplantlets cultivated in a stirred tank bioreactor ultimately assumed compact spherical shape, presumably to minimize exposure to hydrodynamic stress.     

Substrate and solvent isotope effects on the fate of the active oxygen species in substrate-modulated reactions of putidamonooxin.

Using 4-methoxybenzoate monooxygenase from Pseudomonas putida, the substrate deuterium isotope effect on product formation and the solvent isotope effect on the stoichiometry of oxygen uptake, NADH oxidation, product and/or H2O2 (D2O2) formation for tight couplers, partial uncouplers, and uncouplers as substrates were measured. These studies revealed for the true, intrinsic substrate deuterium isotope effect on the oxygenation reaction a k1H/k2H ratio of < 2.0, derived from the inter- and intramolecular substrate isotope effects. This value favours a concerted oxygenation mechanism of the substrate. Deuterium substitution in a tightly coupling substrate initiated a partial uncoupling of oxygen reduction and substrate oxygenation, with release of H2O2 corresponding to 20% of the overall oxygen uptake. This H2O2 (D2O2) formation (oxidase reaction) almost completely disappeared when the oxygenase function was increased by deuterium substitution in the solvent. The electron transfer from NADH to oxygen, however, was not affected by deuterium substitution in the substrate and/or the solvent. With 4-trifluoromethylbenzoate as uncoupling substrate and D2O as solvent, a reduction (peroxidase reaction) of the active oxygen complex was initiated in consequence of its extended lifetime. These additional two electron-transfer reactions to the active oxygen complex were accompanied by a decrease of both NADH oxidation and oxygen uptake rates. These findings lead to the following conclusions: (a) under tightly coupling conditions the rate-limiting step must be the formation time and lifetime of an active transient intermediate within the ternary complex iron/peroxo/substrate, rather than an oxygenative attack on a suitable C-H bond or electron transfer from NADH to oxygen. Water is released after the monooxygenation reaction; (b) under uncoupling conditions there is competition in the detoxification of the active oxygen complex between its protonation (deuteronation), with formation of H2O2 (D2O2) and its further reduction to water. The additional two electron-transfer reactions onto the active oxygen complex then become rate limiting for the oxygen uptake rate.     

Ligand binding equilibrium and kinetic measurements on the dimeric myoglobin of Busycon canaliculatum and the comparative ligand binding of diverse non-cooperative heme proteins

The rate constants and delta H degrees for the non-cooperative dimeric Busycon myoglobin are: oxygen, k' = 4.75 X 10(7) M-1 sec-1, k = 71 sec-1, and CO, l'= 3.46 X 10(5) M-1 sec-1, l = 0.0052 sec-1 at 20 degrees C, pH 7, delta H degrees = -3 kcal/mol for O2 and CO.2. Log-log plots of k vs K for oxygen and of l' vs L for CO binding for numerous non-cooperative hemoglobins and myoglobins point to a large steric influence of the protein on heme ligation reactions. Many of the proteins behave as "R" state for one ligand, but "T" for the other.     

Evidence that bicarbonate is not the substrate in photosynthetic oxygen evolution

It is widely accepted that the oxygen produced by photosystem II of cyanobacteria, algae, and plants is derived from water. Earlier proposals that bicarbonate may serve as substrate or catalytic intermediate are almost forgotten, though not rigorously disproved. These latter proposals imply that CO2 is an intermediate product of oxygen production in addition to O2. In this work, we investigated this possible role of exchangeable HCO3- in oxygen evolution in two independent ways. (1) We studied a possible product inhibition of the electron transfer into the catalytic Mn4Ca complex during the oxygen-evolving reaction by greatly increasing the pressure of CO2. This was monitored by absorption transients in the near UV. We found that a 3,000-fold increase of the CO2 pressure over ambient conditions did not affect the UV transient, whereas the S(3) --> S(4) --> S(0) transition was half-inhibited by raising the O2 pressure only 10-fold over ambient, as previously established. (2) The flash-induced O2 and CO2 production by photosystem II was followed simultaneously with membrane inlet mass spectrometry under approximately 15% H2(18)O enrichment. Light flashes that revealed the known oscillatory O2 release failed to produce any oscillatory CO2 signal. Both types of results exclude that exchangeable bicarbonate is the substrate for (and CO2 an intermediate product of) oxygen evolution by photosynthesis. The possibility that a tightly bound carbonate or bicarbonate is a cofactor of photosynthetic water oxidation has remained.